NZ560713A

NZ560713A - VEGF antagonist formulations

Info

Publication number: NZ560713A
Application number: NZ560713A
Authority: NZ
Inventors: Daniel Dix; Kelly Frye; Susan Kautz
Original assignee: Regeneron Pharma
Priority date: 2005-03-25
Filing date: 2006-03-22
Publication date: 2010-11-26
Also published as: CA2598711A1; US20120178683A1; US11806398B2; WO2006104852A3; AU2006229930C1; PT2586459T; US20190388539A1; JP2013079270A; LT2586459T; WO2006104852A2; US20150079087A1; US9511140B2; JP5376573B2; IL185606A; EP3195874A1; US9416167B2; HK1116050A1; ES2633574T3; US8710004B2; CY1119243T1

Abstract

Provided is a stable liquid formulation of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist, comprising 10-50 mg/ml of a fusion protein having a receptor component consisting of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, wherein the first VEGF receptor is FltI and the second VEGF receptor is FlkI or Flt4, and a multimerizing component, a buffer, 25-150 mM NaCl, 5-30% sucrose, 0.05-0.10% polysorbate, at a pH of about 6- 6.5, wherein at least about 92 percent of the fusion protein maintains its native conformation after 12 months of storage at 5°C. The composition can be also in (pre)lyophilized form.

Description

New Zealand Paient Spedficaiion for Paient Number 560713 VEGF ANTAGONIST FORMULATIONS Field of the Invention id="p-1" id="p-1" id="p-1"

[0001] The present invention is directed to pharmaceutical formulations comprising agents capable of inhibiting vascular endothelial growth factor (VEGF), and to methods for making and using such formulations. The invention includes pharmaceutical formulations having increased stability.

Statement of Related Art id="p-2" id="p-2" id="p-2"

[0002] Vascular endothelial growth factor (VEGF) expression is nearly ubiquitous in human cancer, consistent with its role as a key mediator of tumor neoangiogenesis. Blockade of VEGF function, by binding to the molecule or its VEGFR-2 receptor, inhibits growth of implanted tumor cells in multiple different xenograft models (see, for example, Gerber et al. (2000) Cancer Res. 60:6253-6258). A soluble VEGF-specific fusion protein antagonist, termed a "VEGF trap" has been described (Kim et al. (2002) Proc. Natl. Acad. Sci. USA 99:11399-404; Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99:11393-8). id="p-3" id="p-3" id="p-3"

[0003] Lyophilization (freeze drying under controlled conditions) is commonly used for long term storage of proteins. The lyophilized protein is substantially resistant to degradation, aggregation, oxidation, and other degenerative processes while in the freeze-dried state (see, for example, U.S. 6,436,897).

BRIEF SUMMARY OF THE INVENTION id="p-4" id="p-4" id="p-4"

[0004] Stable formulations of a VEGF-specific fusion protein antagonist are herein provided. The pharmaceutical^ acceptable formulations of the invention comprise the VEGF "trap" antagonist with a pharmaceutically acceptable carrier. In specific embodiments, liquid and freeze-dried, or lyophilized formulations are provided. id="p-5" id="p-5" id="p-5"

[0005] In a first aspect, the invention features a stable liquid formulation of a VEGF-specific fusion protein antagonist, comprising a fusion protein comprising a receptor component consisting essentially of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, and a multimerizing component, one or more buffers, and one or more thermal stabilizers. In a specific embodiment of the VEGF-specific fusion protein antagonist, the first VEGF receptor is Flt1 and the second VEGF receptor is Flk1 or Flt4. In a more specific embodiment the fusion protein has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, the buffer is a phosphate buffer and/or citrate. More preferably, the buffers are phosphate and citrate. In one embodiment, the thermal stabilizers are NaCI and/or sucrose. More preferably, the thermal stabilizers are both NaCI and sucrose. id="p-6" id="p-6" id="p-6"

[0006] In a specific embodiment, the stable liquid formulation of a VEGF-specific fusion protein antagonist comprises 1-10 mM phosphate buffer, 1-10 mM citrate, 25-150 mM NaCI, 5-30% 1 WO 2006/104852 PCT/US2006/010600 sucrose, 10-50 mg/ml of the fusion protein, at a pH of about 6-6.5. In a more specific embodiment, the stable liquid formulation comprises 5 mM phosphate buffer, 5 mM citrate buffer, 100 mM NaCI, 20% sucrose, 25 mg/ml of the fusion protein, at a pH of about 6.0. Additionally, polysorbate may be present, for example 0.05-0.15% polysorbate 20. The stable liquid formulation of the VEGF-specific fusion protein antagonist of the invention exhibits little or no precipitation after storage of a 25 mg/ml VEGF formulation for about 6 months at -80°C and little or no precipitation after storage for 6 months at 5°C. id="p-7" id="p-7" id="p-7"

[0007] In a second aspect, the invention features a high concentration stable liquid formulation of a VEGF antagonist comprising 1-50 mM histidine, 25-150 mM NaCI, 5-30% sucrose, 50-100 mg/ml of the fusion protein, at a pH of about 6-6.5, and either 0.1-0.5% polysorbate or 1-5% PEG. In a more specific embodiment, the high concentration stable liquid formulation comprises 10 mM histidine, 50 mM NaCI, 5-20% sucrose, 50-100 mg/ml of the fusion protein, at a pH of about 6.0-6.5, with either 0.1% polysorbate (e.g., polysorbate 20) or 3% PEG (e.g., PEG 3350). The high concentration stable liquid formulation of the VEGF-specific fusion protein antagonist of the invention exhibits less than about 3% degradation after 15 months of storage at 5°C (75 or 100 mg/ml VEGF trap protein) or less than about 1.5% degradation after 24 months (50 mg/ml). id="p-9" id="p-9" id="p-9"

[0009] In a third aspect, the invention features a pre-lyophilized formulation of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist, comprising a (i) fusion protein comprising a receptor component consisting essentially of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, and a multimerizing component, (ii) a buffer, (iii) an organic co-solvent or bulking agent, and (iv) one or more lyoprotectants. In various embodiments, the buffer is histidine, the organic co-solvent or bulking agent is PEG, and the lyoprotectant(s) is at least one of glycine and sucrose. In one embodiment, the pre-lyophilized formulation of the invention does not contain a preservative. id="p-9" id="p-9" id="p-9"

[0009] In one embodiment of the pre-lyophilized formulation of the invention, the formulation comprises 5-50 mM histidine, 0.1-3.0% PEG, 0.25-3.0% glycine, 0.5-6.0% sucrose, and 5-75 mg/ml of the fusion protein, at a pH of about 6.0- 6.5. In any embodiment, the pre-lyophilized formulation may further comprise up to 0.05 mM citrate and/or 0.003-0.005% polysorbate. The polysorbate present may be, for example, polysorbate 20. id="p-10" id="p-10" id="p-10"

[0010] In a more specific embodiment, the pre-lyophilized formulation comprises about 10 mM histidine, about 1.5% PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 12.5 to 75 mg/ml VEGF-specific fusion protein, at a pH of about 6.25. In specific embodiments, the fusion protein comprises the protein sequence of SEQ ID NO:4, present as a multimer, e.g., a dimer. In separate embodiments, the reconstituted formulation is 2 times the concentration of the pre-lyophilized formulation, e.g., a 20 mg fusion protein/ml pre-lyophilized formulation is reconstituted to a final formulation of 60 mg fusion protein/ml. Generally, the lyophilized 2 RECEIVED at IPONZ on 22 September 2010 formulation is reconstituted with sterile water suitable for injection. In one embodiment, the reconstitution liquid may be bacteriostatic water. id="p-11" id="p-11" id="p-11"

[0011] In a preferred embodiment, the pre-lyophilized formulation consists essentially of about 10 mM histidine, about 1.5% PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 50 mg/ml of the fusion protein having the sequence of SEQ ID NO:4 as a dimer, at a pH of about 6.25. Citrate (less than or equal to about 0.02 mM) and/or polysorbate (less than or equal to about 0.0005%) may be present. Optionally, the pre-lyophilized formulation does not contain a preservative, a phosphate buffer, and/or more than trace amounts of NaCI. In one embodiment, the pre-lyophilized formulation consists of about 10 mM histidine, about 1.5% 10 PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 50 mg/ml of the VEGF trap protein (SEQ ID NO:4), pH 6.3, and upon reconstitution contains 20 mM histidine, 3% PEG, 1.5% glycine, about 5% sucrose, and about 100 mg/ml VEGF trap protein. id="p-12" id="p-12" id="p-12"

[0012] In a fourth aspect, the invention features a method of producing a lyophilized formulation of a VEGF-specific fusion protein antagonist, comprising subjecting the pre- lyophilized formulation of the invention to lyophilization to generate a lyophilized formulation. The lyophilized formulation may be lyophilized by any method known in the art for lyophilizing a liquid. id="p-13" id="p-13" id="p-13"

[0013] In a fifth related aspect, the invention features a method of producing a reconstituted lyophilized formulation of a VEGF-specific fusion protein antagonist, comprising reconstituting the lyophilized formulation of the invention to a reconstituted formulation. In one embodiment, the reconstituted formulation is twice the concentration of the pre-lyophilized formulation, e.g., the method of the invention comprises: (a) producing a pre-lyophilized formulation of a VEGF-specific fusion protein antagonist, (b) subjecting the pre-lyophilized formulation of step (a) to lyophilization; and (c) reconstituting the lyophilized formulation of step (b). id="p-14" id="p-14"

[0014] In specific embodiments of the method of producing a reconstituted lyophilized formulation, a pre-lyophilized solution is present in a vial as a 25 mg VEGF-specific fusion protein antagonist per ml solution of pre-lyophilized formulation, which is lyophilized and reconstituted to an 50 mg/ml solution. In another embodiment, a 30 mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 60 mg/ml solution. In another embodiment, a 40 30 mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 80 mg/ml solution. In another embodiment, a 12.5 mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 25 mg/ml solution. In another embodiment, a 50 mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 100 mg/ml solution. In another embodiment, a 75 mg/ml pre-lyophilized solution is lyophilized and reconstituted to a 150 mg/ml solution. Preferably, the reconstituted 35 lyophilized formulation does not contain a preservative. [0014a] In a further related aspect, the invention features a stable liquid formulation of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist, comprising 10-50 mg/ml of a fusion protein having a receptor component consisting of an immunoglobulin-like 3 RECEIVED at IPONZ on 28 October 2010 (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, wherein the first VEGF receptor is Flt1 and the second VEGF receptor is Flk1 or Flt4, and a multimerizing component, a buffer, 25-150 mM NaCI, 5-30% sucrose, 0.05-0.10% polysorbate, at a pH of about 6- 6.5, wherein at least about 92 percent of the fusion protein maintains its 5 native conformation after 12 months of storage at 5°C. [0014b] In a further related aspect, the invention features a pre-lyophilized formulation, comprising a vascular endothelial growth factor (VEGF)- specific fusion protein antagonist having the amino acid sequence of SEQ ID NO:4, 5-50 mM histidine, 0.1-3.0% PEG 3350, and 0.25-3.0% glycine, at a pH of about 6.0- 6.5, wherein at least about 92 percent of the fusion 10 protein maintains its native conformation after 12 months of storage at 5°C. [0014c] In a further related aspect, the invention features a liquid stable formulation of a VEGF antagonist, comprising 1-50 mM histidine, 25-150 mM NaCI, 5-30% sucrose, 0.01-0.5% polysorbate, and 50-100 mg/ml of a fusion protein having a receptor component consisting of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second 15 VEGF receptor, wherein the first VEGF receptor is Fltl and the second VEGF receptor is Flkl or Flt4, and a multimerizing component, at a pH of about 6.0- 6.5, and optionally comprising 0.1-5% PEG, wherein at least about 92 percent of the fusion protein maintains its native conformation after 12 months of storage at 5°C. id="p-15" id="p-15" id="p-15"

[0015] Other aspects and advantages will become apparent from a review of the ensuing 20 detailed description. 3a WO 2006/104852 PCT/US2006/010600 DETAILED DESCRIPTION OF THE INVENTION id="p-16" id="p-16" id="p-16"

[0016] The present invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting unless indicated, since the scope of the present invention will be limited only by the appended claims. id="p-17" id="p-17" id="p-17"

[0017] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "a method" include one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

General Description id="p-18" id="p-18" id="p-18"

[0018] Safe handling and administration of formulations comprising proteins represent significant challenges to pharmaceutical formulators. Proteins possess unique chemical and physical properties that present stability problems: a variety of degradation pathways exist for proteins, implicating both chemical and physical instability. Chemical instability includes deamination, aggregation, clipping of the peptide backbone, and oxidation of methionine residues. Physical instability encompasses many phenomena, including, for example, aggregation. id="p-19" id="p-19" id="p-19"

[0019] Chemical and physical stability can be promoted by removing water from the protein. Lyophilization (freeze-drying under controlled conditions) is commonly used for long-term storage of proteins. The lyophilized protein is substantially resistant to degradation, aggregation, oxidation, and other degenerative processes while in the freeze-dried state. The lyophilized protein is normally reconstituted with water optionally containing a bacteriostatic preservative (e.g., benzyl alcohol) prior to administration.

Definitions id="p-20" id="p-20" id="p-20"

[0020] The term "carrier" includes a diluent, adjuvant, excipient, or vehicle with which a composition is administered. Carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. id="p-21" id="p-21" id="p-21"

[0021] The term "excipient" includes a non-therapeutic agent added to a pharmaceutical composition to provide a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. id="p-22" id="p-22" id="p-22"

[0022] The term "lyophilized" or "freeze-dried" includes a state of a substance that has been 4 subjected to a drying procedure such as lyophilization, where at least 50% of moisture has been removed. id="p-23" id="p-23" id="p-23"

[0023] The phrase "bulking agent" includes a compound that is pharmaceutical^ acceptable and that adds bulk to a lyo cake. Generally, acceptable bulking agents known to the art include, for example, carbohydrates, including simple sugars such as dextrose, ribose, fructose and the like, alcohol sugars such as mannitol, inositol and sorbitol, disaccharides including trehalose, sucrose and lactose, naturally occurring polymers such as starch, dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin), glycogen, and synthetic monomers and polymers. In the formulations of the invention, PEG 3350 is an organic co-solvent which is used to stabilize the fusion protein when agitated, mixed, or handled, and as a bulking agent to help produce an acceptable bulk. id="p-24" id="p-24" id="p-24"

[0024] The term "lyoprotectant" includes a substance that may be added to a freeze-dried or lyophilized formulation to help maintain protein structure when freeze-dried or lyophilized. id="p-25" id="p-25" id="p-25"

[0025] A "preservative" includes a bacteriostatic, bacteriocidal, fungistatic or fungicidal compound that is generally added to formulations to retard or eliminate growth of bacteria or other contaminating microorganisms in the formulations. Preservatives include, for example, benzyl alcohol, phenol, benzalkonium chloride, m-cresol, thimerosol, chlorobutanol, methylparaben, propylparaben and the like. Other examples of pharmaceutically acceptable preservatives can be found in the USP.

VEGF Antagonists id="p-26" id="p-26" id="p-26"

[0026] An VEGF antagonist is a compound capable of blocking or inhibiting the biological action of vascular endothelial growth factor (VEGF), and includes fusion proteins capable of trapping VEGF. In a preferred embodiment, the VEGF antagonist is the fusion protein of SEQ ID NO:2 or 4; more preferably, SEQ ID NO:4. In specific embodiments, the VEGF antagonist is expressed in a mammalian cell line such as a CHO cell and may be modified post-translationally. In a specific embodiment, the fusion protein comprises amino acids 27-457 of SEQ ID NO:4 and is glycosylated at Asn residues 62, 94, 149, 222 and 308. id="p-27" id="p-27" id="p-27"

[0027] The VEGF antagonist of the methods and formulations of the invention can be prepared by any suitable method known in the art, or that comes to be known. The VEGF antagonist is preferably substantially free of protein contaminants at the time it is used to prepare the pharmaceutically acceptable formulation. By "substantially free of protein contaminants" is meant, preferably, that at least 90 % of the weight of protein of the VEGF-specific fusion protein antagonist preparation used for making a formulation is VEGF fusion protein antagonist protein, more preferably at least 95%, most preferably at least 99%. The fusion protein is preferably substantially free of.aggregates. "Substantially free of aggregates" means that at least 90% of the weight of fusion protein is not present in an aggregate at the time the fusion protein is used to prepare the pharmaceutically effective formulation. The fusion protein of the methods and WO 2006/104852 PCT/US2006/010600 formulations of the invention may contain low or trace amounts of compounds as a results of the purification process, for example, low or trace amounts of citrate and/or polysorbate. In one embodiment of the pre-lyophilized formulation of the invention containing about 50 mg of fusion protein/ml, citrate may be present at a concentration of about 0.02 mM and/or polysorbate may be present at a concentration of about 0.0005%. If the pre-lyophilized formulation is reconstituted after lyophilization to half of the original volume (e.g., 100 mg/ml of fusion protein), the resulting concentrations may be 0.04 mM citrate and/or 0.001% polysorbate.

Lyophilization and Lyophilized Formulations id="p-28" id="p-28" id="p-28"

[0028] In one aspect of the invention, a pharmaceutically acceptable formulation comprising a VEGF-specific fusion protein antagonist is provided, wherein the formulation is a freeze-dried or lyophilized formulation. Lyophilized formulations can be reconstituted into solutions, suspensions, emulsions, or any other suitable form for administration or use. Lyophilized formulations are typically first prepared as liquids, then frozen and lyophilized. The total liquid volume before lyophilization can be less, equal to, or more than, the final reconstituted volume of the lyophilized formulation. The lyophilization process is well known to those of ordinary skill in the art, and typically includes sublimation of water from a frozen formulation under controlled conditions. id="p-29" id="p-29" id="p-29"

[0029] Lyophilized formulations can be stored at a wide range of temperatures. Lyophilized formulations may be stored below 25°C, for example, refrigerated at 4°C, or at room temperature (e.g., approximately 25°C). Preferably, lyophilized formulations are stored below about 25°C, more preferably, at about 4-20°C; below about 4°C; below about -20°C; about -40°C; about -70°C, or about -80°C. id="p-30" id="p-30" id="p-30"

[0030] Lyophilized formulations are typically reconstituted for use by addition of an aqueous solution to dissolve the lyophilized formulation. A wide variety of aqueous solutions can be used to reconstitute a lyophilized formulation. Preferably, lyophilized formulations are reconstituted using water. Lyophilized formulations are preferably reconstituted with a solution consisting essentially of water (e.g., USP WFI, or water for injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl alcohol). However, solutions comprising buffers and/or excipients and/or one or more pharmaceutically acceptable carries can also be used. id="p-31" id="p-31" id="p-31"

[0031] Freeze-dried or lyophilized formulations are typically prepared from liquids, that is, from solutions, suspensions, emulsions, and the like. Thus, the liquid that is to undergo freeze-drying or lyophilization preferably comprises all components desired in a final reconstituted liquid formulation. As a result, when reconstituted, the freeze-dried or lyophilized formulation will render a desired liquid formulation upon reconstitution. A preferred liquid formulation used to generate a freeze-dried or lyophilized formulation comprises a VEGF-specific fusion protein antagonist in a pharmaceutically effective amount, a buffer, a stabilizer, and a bulking agent.

Freeze-dried or lyophilized formulations preferably comprise histidine, since histidine, in 6 comparison to phosphate, is more effective at stabilizing the fusion protein when the fusion protein is lyophilized. Organic co-solvents, such as PEG 3350, are used to stabilize the fusion protein when agitated, mixed, or handled. A lyoprotectant is preferably used in freeze-dried or lyophilized formulations. Lyoprotectants help to maintain the secondary structure of proteins when freeze-dried or lyophilized. Two preferred example lyoprotectants are glycine and sucrose, which are preferably used together.

Stable Liquid Formulations id="p-32" id="p-32" id="p-32"

[0032] In one aspect, the invention provides a stable pharmaceutically acceptable formulation comprising a VEGF-specific fusion protein antagonist, wherein the formulation is a liquid formulation. Preferably, the liquid formulation comprises a pharmaceutically effective amount of the fusion protein. The formulation can also comprise one or more pharmaceutically acceptable carriers, buffers, bulking agents, stabilizers, preservatives, and/or excipients. An example of a pharmaceutically acceptable liquid formulation comprises a VEGF-specific fusion protein antagonist in a pharmaceutically effective amount, a buffer, a co-solvent, and one or more stabilizers. id="p-33" id="p-33" id="p-33"

[0033] A preferred liquid formulation comprises phosphate buffer, an organic co-solvent, and one or more thermal stabilizers to minimize formation of aggregates and low molecular weight products when stored, and about 10 mg/ml to about 50 mg/ml fusion protein, wherein the formulation is from about pH 6.0-6.5. A preferred liquid formulation comprises about 5 mM phosphate buffer, about 5 mM citrate, about 100 mM NaCI, about 25% sucrose, and about 10-50 mg/ml fusion protein, wherein the formulation is at a pH of about 6.0; optionally polysorbate may be present (e.g., 0.1% polysorbate 20). Although either NaCI or sucrose can be used as a stabilizer, a combination of NaCI and sucrose has been established to stabilize the fusion protein more effectively than either individual stabilizer alone. id="p-34" id="p-34" id="p-34"

[0034] Stability is determined in a number of ways at specified time points, including determination of pH, visual inspection of color and appearance, determination of total protein content by methods known in the art, e.g., UV spectroscopy, SDS-PAGE, size-exclusion HPLC, bioassay determination of activity, isoelectric focusing, and isoaspartate quantification. In one example of a bioassay useful for determining VEGF antagonist activity, a BAF/3 VEGFR1/EPOR cell line is used to determine VEGF165 binding by the VEGF-specific fusion protein antagonist of the invention. id="p-35" id="p-35" id="p-35"

[0035] Formulations, whether liquid or freeze-dried and lyophilized, can be stored in an oxygen-deprived environment. Oxygen-deprived environments can be generated by storing the formulations under an inert gas such as, for example, argon, nitrogen, or helium. 7 WO 2006/104852 PCT/US2006/010600 EXAMPLES id="p-36" id="p-36" id="p-36"

[0036] Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only to the appended claims. id="p-37" id="p-37" id="p-37"

[0037] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. id="p-38" id="p-38" id="p-38"

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Example 1. Stability of a 50 mg/ml Liquid Formulation of VEGF Trap id="p-39" id="p-39" id="p-39"

[0039] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 0.1% polysorbate 20, 20% sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 3, 6, 9, 12, 18 and 24 months. Stability was determined by SE-HPLC. The results, shown in Table 1, show that 98.6% and 98.3% of VEGF trap protein remained intact (non-degraded) at 12 and 24 months, respectively. Turbidity was measured at OD4o5 nm; and percent recovered protein by size exclusion HPLC.

Table 1. Stability of 50 mg/ml VEGF Trap Protein When Stored at 5 °C (VGFT-SS065) Months Visual Appearance Turbidity pH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass 0.00 6.2 102 98.8 6 Pass 0.01 6.2 103 98.7 9 Pass 0.01 6.3 102 98.2 12 Pass 0.01 6.3 106 98.6 18 Pass 0.00 6.3 103 98.4 24 Pass 0.00 6.2 93 98.3 id="p-40" id="p-40" id="p-40"

[0040] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 3% PEG 3350, 20% 8 WO 2006/104852 PCT/US2006/010600 sucrose, and 50 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 3, 6, 9, 12, 18 and 24 months. Stability results are shown in Table 2.

Table 2. Stability of 50 mg/ml VEGF Trap Protein When Stored at 5 °C (VGFT-SS065) Months Visual Appearance Turbidity pH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.2 100 99.0 3 Pass 0.00 6.2 100 98.8 6 Pass 0.01 6.3 103 98.5 9 Pass 0.00 6.3 103 98.3 12 Pass 0.01 6.3 110 98.3 18 Pass 0.00 6.3 113 98.0 24 Pass 0.01 6.2 90 97.8 Example 2. Stability of a 75 mg/ml Liquid Formulation of VEGF Trap [0041] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 0.1% polysorbate 20, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in Table 3.

Table 3. Stability of 75 mg/ml VEGF Trap Protein When Stored at 5 °C (VGFT-SS101) Months Visual Appearance T urbidity pH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.2 100 97.1 1 Pass 0.00 6.2 96 97.0 2.3 Pass 0.00 6.2 98 96.7 3 Pass 0.00 6.2 97 96.1 9 Pass -0.01 6.0 101 96.0 12 Pass 0.00 6.3 110 94.5 Pass 0.00 6.3 92 95.6 id="p-42" id="p-42" id="p-42"

[0042] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 3% PEG 3350, 20% sucrose, and 75 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in Table 4. 9 WO 2006/104852 PCT/US2006/010600 Table 4. Stability of 75 mg/ml VEGF Trap Protein When Stored at 5 °C (VGFT-SS101) Months Visual Appearance Turbidity pH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.2 100 96.8 1 Pass 0.00 6.2 99 96.7 2.3 Pass 0.00 6.2 97 96.3 3 Pass 0.00 6.2 89 95.6 9 Pass -0.01 6.2 98 95.4 12 Pass -0.01 6.3 112 94.1 Pass 0.00 6.3 98 94.8 Example 3. Stability of a 100 mg/ml Liquid Formulation of VEGF Trap [0043] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 0.1% polysorbate 20, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in Table 5.

Table 5. Stability of 100 mg/ml VEGF Trap Protein Stored at 5 °C (VGFT-SS101) Months Visual Appearance Turbidity pH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.3 100 96.7 1 Pass 0.00 6.2 92 96.6 2.3 Pass 0.00 6.2 92 96.2 6 Pass 0.00 6.2 99 95.5 9 Pass -0.01 6.2 92 95.5 12 Pass -0.01 6.2 110 93.9 Pass 0.00 6.3 108 94.8 id="p-44" id="p-44" id="p-44"

[0044] A liquid formulation containing 10 mM phosphate, 50 mM NaCI, 3% PEG 3350, 20% sucrose, and 100 mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, was stored at 5 °C and samples tested at 0,1, 2.3, 3, 9, 12 and 15 months. Stability results are shown in Table 6.

WO 2006/104852 PCT/US2006/010600 Table 6. Stability of 100 mg/ml VEGF Trap Protein Stored at 5 °C (VGFT-SS101) Months Visual Appearance Turbidity PH % VEGF Trap Recovered % VEGF Trap Native Configuration 0 Pass 0.00 6.3 100 96.5 1 Pass 0.01 6.2 94 96.2 2.3 Pass 0.01 6.2 93 95.7 6 Pass 0.01 6.2 102 94.6 9 Pass 0.00 6.2 95 94.6 12 Pass 0.00 6.3 96 92.8 Pass 0.01 6.3 102 93.9 Example 4. Further Embodiments of Stable VEGF Trap Formulations id="p-45" id="p-45" id="p-45"

[0045] In one embodiment, the invention provides a stable liquid VEGF-binding fusion protein (VEGF trap) formulations comprising 5 mM phosphate, 5 mM citrate, 100 mM NaCI, 0.1% Polysorbate 20, 20% sucrose, 25 mg/ml VEGF trap protein, pH 6.0. This formulation can either be delivered subcutaneously or diluted and delivered by intravenous infusion. Due to the high osmolality of this formulation, it is diluted 3-fold to achieve an iso-osmolar solution for intravenous administration. Stability studies showed less than about 1 % degradation was detected after 3 years of storage at 2-8°C. id="p-46" id="p-46" id="p-46"

[0046] In one embodiment, the invention features a lyophilized formulation which is preferably concentrated two-fold from the pre-lyophilized to the post-lyophilized formulation, e.g., 50 to 100 mg/ml; 75 to 150 mg/ml, or 100 to 200 mg/ml VEGF trap protein. In one specific embodiment, the pre-lyophilized formulation comprises 10 mM histidine, 1.5% PEG 3350, 0.75% glycine, 2.5% sucrose, 50 mg/ml VEGF trap protein, pH 6.3, and is reconstituted to a formulation comprising 20 mM histidine, 3% PEG 3350, 1.5% glycine, 5% sucrose, 100 mg/ml VEGF trap protein, pH 6.3. Stability studied showed no degradation of the VEGF trap was detected after 6 months of storage at 2-8 °C. id="p-47" id="p-47" id="p-47"

[0047] In one embodiment of a liquid formulation, the formulation comprises 10 mM histidine, 50 mM NaCI, 5-20% sucrose, 50-100 mg/ml VEGF trap, and one of 0.1% polysorbate 20 or 3% PEG 3350. One advantage of this liquid formulation is that it provides a higher concentration of VEGF trap without requiring the manufacture of a lyophilized product. Thus, this formulation provides ease for subcutaneous delivery, for example, by allowing provision of a liquid pre-filled syringe at a concentration higher than that delivered by IV infusion. Also, this formulation could advantageously be used to provide lower infusion volumes and shorter infusion times. The amount of degradation determined by SE-HPLC following incubation at 5 °C for up to 15 or 24 months is summarized in Table 7. 11 WO 2006/104852 PCT/US2006/010600 Table 7. Stability of Liquid Formulation with 50-100 mg/ml VEGF Trap (VGFT-SS101) Incubation (months) VEGF Trap % Polysorbate 20 (mg/ml) % PEG 3350 % Degradation 24 50 0.1 - 0.7 24 50 - 3 1.3 75 0.1 - 1.5 75 - 3 2.0 100 0.1 - 1.9 100 - 3 2.6 Example 5. Stability and Activity of Lyophilized and Liquid id="p-48" id="p-48" id="p-48"

[0048] The stability of a reconstituted lyophilized formulation was determined over a 6 month period. The pre-lyophilized formulation contained 10 mM histidine, 1.5% PEG 3350, 2.5% sucrose, 0.75% glycine and 50 mg/ml VEGF trap protein. After lyophilization, the reconstituted formulation contained 20 mM histidine, 3% PEG 3350, 5% sucrose, 1.5% glycine, and 100 mg/ml VEGF trap protein (SEQ ID NO:4). The results shown in Table 8. Activity was determined in a cell based bioassay which directly measures the ability of the VEGF trap to inhibit the biological effects of human VEGF on a mouse Baf/3 VEGFR1/EpoR cell line. Therefore, this bioassay directly measures the biological activity of the protein. The results are expresses as percent relative potency (test sample IC50/ reference VEGF IC5o standard x 100). The binding affinity of VEGF to the VEGF trap is measured using a sensitive ELISAthat specifically measures free VEGF in equilibrated mixtures containing VEGF and various concentrations of the VEGF trap. Results are expressed as percent relative binding (IC50 of test sample/IC50 of reference x 100). Measured pH ranged between 6.3 - 6.5. All solutions where visually clear. The concentration of VEGF trap recovered was determined with a UV spectrophotometer as mg/ml at A28o nm. The percent VEGF trap recovered in the native configuration (main peak purity) was determined with SE-HPLC.

Table 8. Stability of VEGF Trap Lyophilized Formulation Stored at 5 °C (VGT-RS475) Months Bioassay Binding Assay % Recovered % Native Configuration 0 120 126 97.9 98.7 1 117 74 97.9 98.6 1 + 24 hr 126 72 99.0 98.5 1+ 4 hr 94 81 101.5 98.2 3 101 98 98.1 98.6 3 + 24 hr 65 94 98.1 98.2 6 + 4 hr 96.9 98.7 6 + 24 hr 98.8 98.6 12 WO 2006/104852 PCT/US2006/010600 id="p-49" id="p-49" id="p-49"

[0049] A formulation containing about 5 mM phosphate, 5 mM citrate, 100 mM NaCI, 0.1% polysorbate 20, 20% sucrose, and 25 mg/ml VEGF trap protein was tested for stability and activity over 36 months when stored at 5 °C. The results are shown in Table 9. All samples were clear and colorless as determined by visual inspection. pH ranged from 6.0-6.1. ^Binding assay results for two measurements (1 and 2 months) are expressed directly and not as a percent of the standard.

Table 9. Stability and Activity of Liquid Formulation (VGT-FS405) Months % Native Configuration Bioassay Binding Assay Protein Content mg/ml 0 99.7 106 72 .0 1 99.9 119 4.4 pM* ;25.2 ;2 ;99.6 ;102 ;5.4 pM* .1 3 99.6 97 88 .1 6 99.6 101 106 .0 9 99.4 89 126 .4 12 99.5 85 95 .2 18 99.4 99 81 .5 24 99.3 75 95 .6 36 98.8 109 79 .6 13

Claims

RECEIVED at IPONZ on 22 September 2010 The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed 5 before the priority date of each claim of this application. Throughout the description and claims of the specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. 13a RECEIVED at IPONZ on 22 September 2010 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A stable liquid formulation of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist, comprising 10-50 mg/ml of a fusion protein having a receptor component 5 consisting of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, wherein the first VEGF receptor is Fltl and the second VEGF receptor is Flkl or Flt4, and a multimerizing component, a buffer, 25-150 mM NaCI, 5-30% sucrose, 0.05-0.10% polysorbate, at a pH of about 6- 6.5, wherein at least about 92 percent of the fusion protein maintains its native conformation after 12 months of storage at 5°C. 10

2. The stable liquid formulation of claim 1, wherein the fusion protein has the amino acid sequence of SEQ ID NO:4.

3. The stable liquid formulation of any one of claims 1-2, wherein the buffer is selected from the group consisting of phosphate, citrate, and a combination thereof.

4. The stable liquid formulation of any one of claims 1-3, wherein the buffer is 1-10 mM 15 phosphate and 1-10 mM citrate.

5. The stable liquid formulation of any one of claims 1-4, comprising 5 mM phosphate buffer, 5 mM citrate buffer, 100 mM NaCI, 20% sucrose, 50 mg/ml of the fusion protein, and 0.1% polysorbate 20, at a pH of about 6.2-6.3.

6. The stable liquid formulation of any one of claims 1-4, comprising 5 mM phosphate buffer, 20 5 mM citrate buffer, 100 mM NaCI, 20% sucrose, and 25 mg/ml of the fusion protein, at a pH of about 6.0.

7. The stable liquid formulation of any one of claims 1-4, comprising 5 mM phosphate buffer, 5 mM citrate buffer, 100 mM NaCI, 25% sucrose, 25 mg/ml of the fusion protein, at a pH of about 6.0. 25

8. A pre-lyophilized formulation, comprising a vascular endothelial growth factor (VEGF)- specific fusion protein antagonist having the amino acid sequence of SEQ ID NO:4, 5-50 mM histidine, 0.1-3.0% PEG 3350, and 0.25-3.0% glycine, at a pH of about 6.0- 6.5, wherein at least about 92 percent of the fusion protein maintains its native conformation after 12 months of storage at 5°C. 30

9. The pre-lyophilized formulation of claim 8, comprising 5-75 mg/ml of the fusion protein. 14 RECEIVED at IPONZ on 28 October 2010

10. The pre-lyophilized formulation of claim 9, comprising about 10 mM histidine, about 1.5% PEG 3350, about 0.75% glycine, about 2.5% sucrose, and about 12.5 to 75 mg/ml VEGF-specific fusion protein, at a pH of about 6.25.

11. The pre-lyophilized formulation of claim 10, wherein the fusion protein is 50 mg/ml. 5

12. A liquid stable formulation of a VEGF antagonist, comprising 1-50 mM histidine, 25-150 mM NaCI, 5-30% sucrose, 0.01-0.5% polysorbate, and 50-100 mg/ml of a fusion protein having a receptor component consisting of an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor and Ig domain 3 of a second VEGF receptor, wherein the first VEGF receptor is Fltl and the second VEGF receptor is Flkl or Flt4, and a multimerizing component, at a pH 10 of about 6.0- 6.5, and optionally comprising 0.1-5% PEG, wherein at least about 92 percent of the fusion protein maintains its native conformation after 12 months of storage at 5°C.

13. The liquid stable formulation of claim 12, comprising about 10 mM histidine, about 50 mM NaCI, 5-20% sucrose, and 0.1% polysorbate 20, at a pH of about 6.0- 6.5, optionally comprising 3% PEG 3350. 15

14. A method of producing a lyophilized formulation of a VEGF-specific fusion protein antagonist, comprising subjecting the pre-lyophilized formulation of any one of claims 8 to11 to lyophilization to generate a lyophilized formulation.

15. A method of producing a reconstituted lyophilized formulation of a VEGF-specific fusion protein antagonist, comprising reconstituting the lyophilized formulation claim 14 with liquid, 20 wherein a reconstituted lyophilized formulation is generated.

16. The method of claim 15, wherein the liquid is sterile water or bacteriostatic water.

17. The stable liquid formulation of claim 1, substantially as hereinbefore described with reference to the Examples.

18. A pre-lyophilized formulation of claim 8, substantially as hereinbefore described with 25 reference to the Examples.

19. A liquid stable formulation of claim 12, substantially as hereinbefore described with reference to the Examples. 15 WO 2006/104852 PCT/US2006/010600 SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> VEGF Antagonist Formulations <130> 4030A-W0 <140> To be assigned <141> 2006-03-22 <150> 60/665,125 <151> 2005-03-25 <160> 4 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 1453 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 1 aagcttgggc tgcaggtcga tcgactctag caaccaccat ggtcagctac tgggacaccg tgcttctcac aggatctagt tccggaggta ccgaaattat acacatgact gaaggaaggg ctaacatcac tgttacttta aaaaagtttc gcataatctg ggacagtaga aagggcttca ggcttctgac ctgtgaagca acagtcaatg atcgacaaac caatacaatc atagatgtgg ctgttggaga aaagcttgtc ttaaattgta acttcaactg ggaataccct tcttcgaagc taaaaaccca gtctgggagt gagatgaaga taacccggag tgaccaagga ttgtacacct agaacagcac atttgtcagg gtccatgaaa caccgtgccc agcacctgaa ctcctggggg ccaaggacac cctcatgatc tcccggaccc gccacgaaga ccctgaggtc aagttcaact ccaagacaaa gccgcgggag gagcagtaca ccgtcctgca ccaggactgg ctgaatggca ccctcccagc ccccatcgag aaaaccatct aggtgtacac cctgccccca tcccgggatg gcctggtcaa aggcttctat cccagcgaca cggagaacaa ctacaagacc acgcctcccg atagcaagct caccgtggac aagagcaggt tgatgcatga ggctctgcac aaccactaca aatgagcggc cgc <210> 2 <211> 458 <212> PRT <213> Artificial Sequence <220> aggatcgatc cccgggcgag ctcgaattcg 60 gggtcctgct gtgcgcgctg ctcagctgtc 12 0 gacctttcgt agagatgtac agtgaaatcc 180 agctcgtcat tccctgccgg gttacgtcac 240 cacttgacac tttgatccct gatggaaaac 30 0 tcatatcaaa tgcaacgtac aaagaaatag 360 ggcatttgta taagacaaac tatctcacac 42 0 ttctgagtcc gtctcatgga attgaactat 480 cagcaagaac tgaactaaat gtggggattg 540 atcagcataa gaaacttgta aaccgagacc 600 aatttttgag caccttaact atagatggtg 660 gtgcagcatc cagtgggctg atgaccaaga 72 0 agggcccggg cgacaaaact cacacatgcc 7 80 gaccgtcagt cttcctcttc cccccaaaac 840 ctgaggtcac atgcgtggtg gtggacgtga 900 ggtacgtgga cggcgtggag gtgcataatg 960 acagcacgta ccgtgtggtc agcgtcctca 1020 aggagtacaa gtgcaaggtc tccaacaaag 1080 ccaaagccaa agggcagccc cgagaaccac 1140 agctgaccaa gaaccaggtc agcctgacct 1200 tcgccgtgga gtgggagagc aatgggcagc 1260 tgctggactc cgacggctcc ttcttcctct 1320 ggcagcaggg gaacgtcttc tcatgctccg 1380 cgcagaagag cctctccctg tctccgggta 1440 1453 1 WO 2006/104852 PCT/US2006/010600 <223> Synthetic <400> 2 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 15 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu 20 25 30 Met Tyr Ser Glu lie Pro Glu lie lie His Met Thr Glu Gly Arg Glu 35 40 45 Leu Val lie Pro Cys Arg Val Thr Ser Pro Asn lie Thr Val Thr Leu 50 55 60 Lys Lys Phe Pro Leu Asp Thr Leu lie Pro Asp Gly Lys Arg lie lie 65 70 75 80 Trp Asp Ser Arg Lys Gly Phe lie lie Ser Asn Ala Thr Tyr Lys Glu 85 90 95 lie Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys 100 105 110 Thr Asn Tyr Leu Thr His Arg Gin Thr Asn Thr He lie Asp Val Val 115 120 125 Leu Ser Pro Ser His Gly lie Glu Leu Ser Val Gly Glu Lys Leu Val 130 135 140 Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly He Asp Phe Asn 145 150 155 160 Trp Glu Tyr Pro Ser Ser Lys His Gin His Lys Lys Leu Val Asn Arg 165 170 175 Asp Leu Lys Thr Gin Ser Gly Ser Glu Met Lys Lys Phe Leu Ser Thr 180 185 190 Leu Thr He Asp Gly Val Thr Arg Ser Asp Gin Gly Leu Tyr Thr Cys 195 200 205 Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val Arg 210 215 220 Val His Glu Lys Gly Pro Gly Asp Lys Thr His Thr Cys Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro He Glu Lys Thr lie Ser Lys Ala Lys Gly 340 345 350 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 2 WO 2006/104852 PCT/US2006/010600 <210> 3 <211> 1377 <212> DWA <213> Artificial Sequence <220> <223> Synthetic <400> 3 atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60 acaggatcta gttccggaag tgataccggt agacctttcg tagagatgta cagtgaaatc 12 0 cccgaaatta tacacatgac tgaaggaagg gagctcgtca ttccctgccg ggttacgtca 180 cctaacatca ctgttacttt aaaaaagttt ccacttgaca ctttgatccc tgatggaaaa 240 cgcataatct gggacagtag aaagggcttc atcatatcaa atgcaacgta caaagaaata 300 gggcttctga cctgtgaagc aacagtcaat gggcatttgt ataagacaaa ctatctcaca 3 60 catcgacaaa ccaatacaat catagatgtg gttctgagtc cgtctcatgg aattgaacta 420 tctgttggag aaaagcttgt cttaaattgt acagcaagaa ctgaactaaa tgtggggatt 480 gacttcaact gggaataccc ttcttcgaag catcagcata agaaacttgt aaaccgagac 540 ctaaaaaccc agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt 600 gtaacccgga gtgaccaagg attgtacacc tgtgcagcat ccagtgggct gatgaccaag 660 aagaacagca catttgtcag ggtccatgaa aaggacaaaa ctcacacatg cccaccgtgc 720 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 780 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 840 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 900 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 960 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1020 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1080 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 1140 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 12 00 aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1260 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1320 gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1377 <210> 4 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu 20 Thr Gly Ser Ser Ser 25 Gly Ser Asp Thr Gly 30 Arg Pro Phe Val Glu Met Tyr Ser Glu lie Pro Glu lie lie His Met Thr Glu 35 40 45 Gly Arg Glu Leu Val lie Pro Cys Arg Val Thr Ser Pro Asn lie Thr 50 55 60 Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu lie Pro Asp Gly Lys 65 70 75 80 Arg lie lie Trp Asp Ser Arg Lys Gly Phe lie He Ser Asn Ala Thr 85 90 95 Tyr Lys Glu lie Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His 100 105 110 Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gin Thr Asn Thr lie lie 115 120 125 Asp Val Val Leu Ser Pro Ser His Gly lie Glu Leu Ser Val Gly Glu 3 WO 2006/104852 PCT/US2006/010600 130 135 140 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly lie 145 150 155 160 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gin His Lys Lys Leu 165 170 175 Val Asn Arg Asp Leu Lys Thr Gin Ser Gly Ser Glu Met Lys Lys Phe 180 185 190 Leu Ser Thr Leu Thr lie Asp Gly Val Thr Arg Ser Asp Gin Gly Leu 195 200 205 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 210 215 220 Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys 225 230 235 240 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 275 280 285 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 290 295 300 Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 305 310 315 320 His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 325 330 335 Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 340 345 350 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 355 360 365 Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 370 375 380 Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 385 390 395 400 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 405 410 415 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 420 425 430 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 4